PROJECT SUMMARY The ?-hemoglobinopathies are the most common genetic blood disorders worldwide, but limited effective treatments have been developed over the last three decades. Induction of normal, but developmentally silenced fetal hemoglobin (HbF) expression reduces anemia and ameliorates clinical severity of sickle cell disease (SCD). Histone deacetylases (HDACs) have been shown to promote silencing of the fetal ?-globin genes in adult erythroid cells, and HDAC inhibitors were shown to increase HbF in patients with ?-hemoglobinopathies. But these had limitations for pharmaceutical application due to the need for intravenous administration and their anti- proliferative effects. This project focuses on creating safe and effective small-molecule oral HbF-inducing agents. In one study, we observed HbF induction with a novel oral conjugate of butyric acid (BA) and ?-aminolevulinate (ALA), named AN-233, and a BACH1 inhibitor that enhanced NRF2 expression in sickle erythroid progenitors and ?-YAC mice. Next, focusing on these small-molecule agents, we will test the central hypothesis that modulation of ?-globin gene transcription through epigenetic histone modifications and enhanced NRF2 binding mediate HbF induction. Specific Aim 1. will test the prediction that the oral prodrug AN-233 induces HbF expression through epigenetic histone modifications and NRF2 activation, using relevant preclinical animal models. Treatments conducted in ?-YAC and SCD transgenic mice will establish the optimal oral dose and schedule of AN233 that induces HbF without producing anti-proliferative effects. The phenotypic effects of AN-233 in SCD mice related to oxidative stress, and its anti-sickling ability will be tested. To achieve an indicator of human drug efficacy we will test AN-233 in anemic juvenile baboon to determine HbF induction and effects on hematopoiesis. We will define mechanisms of ?-globin activation by AN-233 involving epigenetic histone acetylation and the role of ALA in heme synthesis and NRF2 activation. Specific Aim 2. Test the prediction that the oral BACH1 inhibitor HPP-D enhances NRF2 binding to the ?-globin promoter ARE to activate transcription. We will establish the BACH1 inhibitor HPP-D, as an HbF inducer using sickle erythroid progenitors and conduct combination drug treatments with clinically relevant agents including hydroxyurea and decitabine. Subsequently, molecular mechanisms of HbF induction by HPP-D through enhanced NRF2 binding in the ?- globin promoter will be evaluated. Finally, the ability of HPP-D to induce HbF expression in the preclinical SCD mice will establish in vivo efficacy. Expected outcomes include establishing specific potent HDAC inhibitors and agents that modulate ?-globin locus chromatin structure in favor of ??globin transcription, for subsequent clinical studies to address the unmet need for improved disease-modifying therapy for SCD.